The prior art has recognized that the presence, or the absence, of certain bile pigments might be used as indicators of the existence of certain disease states. For example, the article, Bile Pigment Fate in Gastrointestinal Tract, Seminars in Hematology, Vol. 9, No. 1 (January) 1972, points out that the presence of fecal bile pigments, which can also be detected in the urine, can be used to help diagnose certain diseases, particularly those of the liver. Such diagnoses are based upon the understanding that urobilinogen is produced in the gut from bilirubin excreted from the liver into the bile; and that when it is diseased, the liver's excretory capacity for urobilinogen is greatly reduced. Consequently, more urobilinogen reaches systemic circulation. It is now generally believed that this, in turn, is due to increased anastomoses between the portal and systemic vessels as well as decreases in the number and function of hepatocytes. There also appears to be evidence that bilirubin and urobilinogen share a mechanism for hepatic uptake. Hence, competition between increased amounts of bilirubin and urobilinogen for hepatic uptake are also thought to contribute to urobilinogenuria associated with hemolytic disorders. In any event, the above noted reference lists the following conditions which may lead to alterations of urinary urobilinogen concentrations:
______________________________________ Factors Influenc- Conditions in Conditions in ing Urinary Uro- Which Urinary Which Urinary bilinogen Urobilinogen Urobilinogen Concentration Tends to Increase Tends to Decrease ______________________________________ Amount of bili- Increased e.g., Decreased e.g., rubin conjugate hemolytic states obstruction of entering gut common bile duct Loss of bile Biliary fistula; pigment from gut small bowel fistula Alterations in Biliary tract Suppression of gut flora infection; gut flora by colonization of antibiotics; most small bowel common in newborn treatment Alteration in Constipation Severe diarrhea transmit time through gut Excretion of Decreased due to urobilinogen hepatocellular by liver disease, trans- hepatic shunting of portal blood; competition with other substances; e.g., bilirubin Renal factors Decrease in urine Decrease glo- volume merular filtra- tion rate; in- crease in urine volume ______________________________________
It is also known that a change in the color of feces is usually present in the case of biliary obstruction and that this color change also is related to urobilinogen concentration. Hence the daily excretion of urobilinogen has been used to assess the degree of obstruction. For example, it is known that the average daily fecal excretion of urobilinogen in normal individuals is about 140 mg (range 40-280 mg day) although there is wide variation in reported values. Males excrete significantly more urobilinogen than females even when expressed on a weight basis (females, typically 0.61 mg/kg body weight/day; males, typically 0.90 mg/kg body weight/day; mean SD). Moreover, these values fluctuate from day to day. However, lower levels (5 mg/24 hour in adults) are found when there is prolonged total obstruction of the biliary tract.
On the other hand, it is also known that in a variety of other disease states there is a surplus rather than a deficit of fecal urobilinogen in relation to the rate of destruction of mature circulating red blood cells and their hemoglobin. This surplus, at times quite large, is represented by what is now generally known as the "early labeled" bile pigment, that fraction of the total bile pigment, bilirubin, or urobilinogen, that exhibits isotopic labeling within a few days after administration of 15.sub.N or 14.sub.C labeled glycine. This is in contrast to the normally much larger fraction (85 to 90%) that exhibits its peak labelling in relation to the destruction of hemoglobin of mature circulating red blood cells in the period of 110 to 130 days after administration of labeled glycine. However, as noted in The Continuing Challenge of Hemoglobin and Bile Pigment Metabolism, Annals of Internal Medicine, Vol. 63, No. 6 (December) 1965, even though there is a presumed relationship of bile bilirubin and fecal urobilinogen to the destruction of hemes, the amount of bilirubin formed from destroyed hemoglobin has not been represented quantitatively in the excreta by recognizable derivatives such as urobilinogen. Among the known pigments (e.g., bile pigments, porphyrins, hemoglobin, indole derivatives, flavins, melanins and pteridins) which may be excreted in human urine there are groups of yellow, brown, and red pigments, generally designated as urochromes, whose chemical structures are still not totally understood. This lack of understanding is due, at least in part, to the highly labile nature of such pigments. Moreover, increased amounts of uroerythrin have been postulated in certain diseases. For example, an article entitled, Isolation and Identification of the Urinary Pigment Uroerythrin, Eur. J. Biochem. 56, 239-244 (1975) teaches: (1) that the red pigment uroerythrin is absorbed by the amorphous urate sediments (sedimentum lateritium), (2) that increased amounts of uroerythrin are observed in patients in certain pathological states (e.g., diseases of the liver) and (3) that uroerythrin most probably has a chemical structure which is based upon a tripyrrole system. This reference does not however correlate the presence of appendicitis with a threshold presence of uroerythrin or any other bile pigment.
Graff, in A Handbook of Routine Urinalysis, J. P. Lippincott Company, (Philadelphia) 1983, cites that large amounts of uroerythrin can occur in acute febrile disease. However, clinical data generated while developing the instant invention includes many cases where patients with appendicitis presented without fever, yet exhibited high levels of uroerythrin (above the threshold of 2.times.10.sup.-4 mg/cc), as confirmed both by instrumental data and induction of the pink to red precipitate described in this disclosure. In addition, many patients without appendicitis, yet having a fever, showed uroerythrin levels below threshold and produced urine samples in which the pink to red precipitate was absent. Clearly this data demonstrates that fever alone is not responsible for creation of high levels of uroerythrin and formation of the pink to red precipitate. Further, the use of pink to red precipitate as an indicator of uroerythrin levels above threshold and thus indicative of appendicitis is valid even under conditions where other red substances are formed in the urine, since all other known disease states causing red coloration present with clinical symptoms different than those in appendicitis, and in most cases have chronic symptomology rather than acute symptomology (e.g., porphyrrhea).
Abasov, in Significance of Bile Pigment Determination in the Urine for the Differential Diagnosis of Appendicitis, Azerb.Med., Zh 0 (8), 1986, p. 11-13, used the presence of certain bile pigments in the urine as an indicator of viral hepatitis, thereby eliminating appendicitis as the disease state in question, but made no correlation between presence of a given pigment and a positive diagnosis of appendicitis.
Uroerythrin has been isolated from human urine and purified as its trimethyl derivative. For example, the previously cited article, Isolation and Identification of the Urinary Pigment Uroerythrin, Eur. J. Biochem, 56 (1975), teaches a purification method based upon introduction of urine into a column of an ion exchange resin such as Amberlite XAD-2 resin which absorb the uroerythrin. Purification is then obtained through conversion of the uroerythrin into its trimethyl derivative and chromatography on silica gel thin-layer plates.